Semisynthetic Pneumococcal Glycoconjugate Nanovaccine

Abstract

Pneumococcal conjugate vaccines offer an excellent safety profile and high protection against the serotypes comprised in the vaccine. However, inclusion of protein antigens fromStreptococcus pneumoniaecombined with potent adjuvants and a suitable delivery system are expected to both extend protection to serotype strains not represented in the formulation and stimulate a broader immune response, thus more effective in young children, elderly, and immunocompromised populations. Along this line, nanoparticle (NP) delivery systems can enhance the immunogenicity of antigens by protecting them from degradation and increasing their uptake by antigen-presenting cells, as well as offering co-delivery with adjuvants. We report herein the encapsulation of a semisynthetic glycoconjugate (GC) composed of a synthetic tetrasaccharide mimicking theS. pneumoniae serotype 14 capsular polysaccharide (CP14) linked to the Pneumococcal surface protein A (PsaA) using chitosan NPs (CNPs). These GC-loaded chitosan nanoparticles (GC-CNPs) were not toxic to human monocyte-derived dendritic cells (MoDCs), showed enhanced uptake, and displayed better immunostimulatory properties in comparison to the naked GC. A comparative study was carried out in mice to evaluate the immune response elicited by the glycoconjugate-administered subcutaneously (SC), where the GC-CNPs displayed 100-fold higher IgG response as compared with the group treated with nonencapsulated GC. Overall, the study demonstrates the potential of this chitosan-based nanovaccine for efficient delivery of glycoconjugate antigens.

Figure 1

Schematic representation of CNP preparation by the ionic gelation method.

Figure 2

Particle size determination by Zetasizer, NP tracking analysis (NTA), and field emission scanning electron microscopy (FESEM) of the blank CNPs and GC-CNPS. (A) Particle size and zeta potential; (B) particle size distribution and the NP concentration of the blank and GC-CNPs, determined by NTA; (C) surface morphology of the blank CNPs and GC-CNPs determined by FESEM and shows that CNPs had a spherical shape; and (D) particle size distribution from the FESEM images calculated using ImageJ software.

Figure 3

CNP cytotoxicity on iDCs. (A) MTS staining assay and (B) 7-AAD assay was performed for both GC-CNPs (blue lines) and blank CNPs (red lines). Results are presented as mean ± SD (n = 4). All the components used in the preparation of CNPs were assayed for the detection of endotoxin activity using the end-point chromogenic limulus amoebocyte assay (LAL test) (Supporting Information and Figure S2).

Figure 4

Assessment of GC-CNP uptake by monocytes using FEG-SEM. (A) Untreated iDCs and (B) iDCs treated with GC-CNPs. The GC-CNPs are pointed with the red arrow marks.

Figure 5

Internalization of Cy5-GC-CNPs (50 μg/million cells) by iDCs at different time points (0.5, 1, 2, and 4 h). The cell membrane is stained with wheat germ agglutinin-488 (WGA-488; green color), the nucleus stained with DAPI (blue color), and the NPs are labeled with Cy5 (red color).

Figure 6

Internalization of the Cy5-GC-CNPs (50 μg/million cells) by MoDCs at 24 h (at 37 and 4 °C). The cell membrane is stained with wheat germ agglutinin-488 (WGA-488; green color), the nucleus stained with DAPI (blue color), and the NPs are labeled with Cy5 (red color).

Figure 7

GC-CNPs induce iDC activation and maturation. The bars in the different colors indicate GC (blue), blank CNPs (red), GC-CNPs (green), and the control with LPS + INF-γ treatment (purple). The data represent the mean ± SD (n = 4).

Figure 8

Antibody response in the mice immunized with PBS, GC, and GC-CNPs 2 weeks after the final immunization. The immunization was performed twice at days 0 and 14 and the serum antibody response in the mice was determined at day 21. The obtained results are represented as anti-PsaA IgG response (A) and anti-CP14 IgG response (B). Statistical difference between the groups is *P < 0.01, **P < 0.001, and ***P < 0.0005. Data represent mean ± SD (n = 6).

Figure 9

Subclass of anti-IgG antibody response in mice immunized with GC-CNPs 2 weeks after the final immunization. The immunization was performed twice at days 0 and 14, and the serum antibody response in the mice was determined at day 21. The obtained results are represented as anti-mPsaA IgG subclass response (A) and anti-CP IgG subclass response (B). Statistical difference between the groups is *P < 0.05, **P < 0.01, and ***P < 0.001. Data represent mean ± SD (n = 6).

Figure 10

Survival of vaccinated mice. Mice were challenged IN by administering 30 cfu (colony forming units) of H₃N₂, followed by 1 × 10⁶ of S. pneumoniae serotype 14. The survival of the mice was monitored for 20 days. The differences between survival rates of six mice per group were analyzed by the Kaplan–Meier survival curve. Log-rank (Mantel–Cox) test P = 0.0714, no significant difference (N = 1).